Portable Fluidic Platform For Rapid Cell-Free Production of Protein Biologics

ABSTRACT

A portable fluidic platform for rapid and flexible end-to-end production of recombinant protein biologics includes a bioreactor system hosting stable and robust cell-free translation systems that is fluidically integrated with modular protein separation functionalities (e.g., size exclusion, ion exchange or affinity chromatography systems) for purification of the cell-free expressed product and which are configurable for process-specific isolation of different proteins, as well as for formulation. The bioreactor utilizes lysates from engineered eukaryotic (e.g., yeast) or prokaryotic (e.g., bacterial) strains that contain factors for protein folding and posttranslational modifications. Combination of various purification modules on the same fluidic platform allows flexibility of re-routing for purification of different proteins depending on specific target requirements. Protein synthesis and purification modules are integrated into self-contained disposable fluidic cartridge that eliminates cross-contamination between runs. The platform allows for flexible production of protein biologics within 24 hours (from DNA to purified product).

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a division of U.S. application Ser. No.15/898,273, filed Feb. 16, 2018, titled “Portable Fluidic Platform ForRapid Cell-Free Production of Protein Biologics,” which is a division ofU.S. application Ser. No. 15/010,528, filed Jan. 29, 2016, titled“Portable Fluidic Platform For Rapid Cell-Free Production of ProteinBiologics,” which claims the benefit of priority to similarly titledU.S. Provisional Application Nos. 62/112,741 and 62/151,086 filed Feb.6, 2015 and Apr. 22, 2015, respectively. All of the above applicationsare incorporated herein by reference in their entireties.

STATEMENT REGARDING GOVERNMENT INTEREST

Certain embodiments were made with Government support under ContractNumber N66001-13-C-4024 awarded by the Defense Advanced ResearchProjects Agency. Accordingly, the United States government may havecertain rights in those certain embodiments.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

A Sequence Listing is being submitted electronically via EFS in the formof a text file, created Jan. 19, 2016, and named“LEID0010UTLseqlist.txt” (6,999 bytes), the contents of which areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The present disclosure relates to a portable platform for production ofprotein biologics. The integrated fluidic platform encompasses robust,cell-free protein synthesis systems coupled to rapid proteinpurification and characterization modules enabling production of proteinbiologics in less than 24 hrs.

BACKGROUND

The safety and efficacy of protein biologics has led to an upswing inthe number of such drugs being introduced into development pipelinestoward clinical use. Additional benefits of protein drugs will berealized by way of their application for specialized needs that includeboth precision and personalized medicine, treatment of orphan diseases,and point-of-care delivery of medical products. Currently, themanufacture of protein biologics is primarily implemented with largescale heterologous expression in bacterial, yeast, and mammaliancell-based systems. Problematically, cell-based gene expression andprotein folding are host-dependent, and optimization for product exportinto the culture fluid is often required to improve production. Otherlimitations of protein production in living cells include: formation ofinsoluble protein aggregates, protein degradation by intracellularproteases, and in some cases, inadequate target expression due to hostcell toxicity of over-expressed protein and/or an inability to conferappropriate humanized post-translational modifications (PTMs) in targetproteins. Overall, such cell-based approaches are impractical forcost-effective and rapid manufacture of low doses of protein biologicsand/or production at the point of need because they require multipleprocesses using large bioreactors, specialized facilities, lengthyproduction cycles, optimum and stable conditions for sustainable cellgrowth, laborious purification protocols, all resulting in longturnaround times between cell transfection and protein isolation as wellas high costs (Spirin 2004, Trends Biotechnol. 22:538-545; Gilbert andAlbala, 2002, Curr. Opin. Chem. Biol. 6:102-105; Mei et al., 2007,Biotechnol. Prog. 23:1305-1311).

In vivo protein expression systems also lack robustness andpredictability due to their lack of modularity and adaptability toproduction at the point of need. For at least these reasons, productionof proteins for such applications will not be cost effective until thereare techniques and systems available to enable production of singledoses or small scale made-to-order products for individual needs thatmeet regulatory criteria for human use.

Cell-free protein synthesis (CFPS) systems have emerged as a powerfulcost-effective technology platform for rapid and efficient production ofpharmaceutical proteins (Goerke and Swartz, 2008, Biotechnol. Bioeng.99:351-367; Kanter et al., 2007, Blood. 109:3393-3399; Yang et al.,2005, Biotechnol. Bioeng. 89:503-511; Yin et al., 2012, MAbs. 4:217-225;Kline et al., 2014, Pharm. Res. 2014, PMID:25511917). CFPS systems havedistinct advantages over in vivo methods for recombinant proteinproduction (Carlson et al., 2012, Biotechnol. Adv. 30:1185-1194; Katzenet al., 2005, Trends Biotechnol. 23:150-156; Swartz 2006, J. Ind.Microbiol. Biotechnol. 33:476-485; Zawada et al., 2011, Biotechnol.Bioeng. 108:1570-1578). Cell-free systems are not constrained byancillary processes required for cell viability and growth (e.g.,homeostatic conditions), thereby allowing optimization of production fora single protein product, as well as optimization of protein complexes,incorporation of non-natural amino acids, high-throughput screening andsynthetic biology. The absence of a cell membrane enables real-timemonitoring, rapid sampling, purification, and direct manipulation of theprotein synthesis process. In addition, the cell-free format avoids theprocess of cell-line generation, thereby allowing system testing andacceleration of the process/product development pipelines.

However, the current eukaryotic CFPS systems suffer from laboriousextract preparation procedures, low and variable product yields,expensive reagents, low protein production rates, small reaction scales,and an unproven track record of expressing complex disulfide bonded orglycosylated proteins.

Recent research advances have led to the production of robust cell-freesystems for protein synthesis in high yields. These advances have beenachieved by increasing reaction duration via continuous supply ofsubstrates and removal of toxic reaction byproducts by diffusionalexchange across a membrane under a continuous exchange cell-free (CECF)format (Shirokov et al., 2007, Methods Mol. Biol. 375:19-55) as well asby activating metabolic networks in vitro for energy production, andimproving extract preparation procedures (Kim and Swartz, 2001,Biotechnol. Bioeng. 74:309-316; Jewett and Swartz, 2004, Biotechnol.Bioeng. 86:19-26; Jewett et al., 2008, Mol. Syst. Biol. 4, DOI:10.1038/msb; Zawada and Swartz, 2005, Biotechnol. Bioeng. 89:407-415;Schoborg et al., 2014, Biotechnol J. 9:630-640; Hodgman and Jewett,2013, Biotechnol. Bioeng. 110:2643-2654). Cell-free systems alsorepresent flexible manufacturing platforms that are highly amenable toautomated liquid handling.

Automated systems for high-throughput protein production andpurification for structural studies have been reported (e.g., Aoki etal., 2009, Protein Expr. Purif. 68:128-136; Makino et al., 2010, MethodsMol. Biol. 607:127-147). Such systems enable rapid dialysis-mode CFPSand purification using immobilized metal affinity chromatography.However, these robotic platforms are currently non-portable and toolarge for point-of-care applications. In addition, use of affinity tagsfor protein purification can lead to products having extra amino acidsnot present in the natural protein sequence (Arnau et al., 2006, ProteinExpr. Purif. 48:1-13). Development of microfluidic array devices forcontinuous-exchange, long-lasting (up to 6 hours) bacterial CFPS havealso been reported (Mei et al., 2007, Biotechnol. Prog. 23:1305-1311;Mei et al., 2010, Lab Chip. 10:2541-2545). In addition,polydimethylsiloxane (PDMS)-based microreactor array chips usingbacterial cell-free extracts and having a disposable reaction chamberchip have been demonstrated to be useful for hosting CFPS reactions(Yamamoto et al., 2008, Anal. Sci. 24:243-246). These microfluidicsystems hold promise for high-throughput protein screening and analysis,but due to their small scale and protein yields are not adequate forproduction and purification of proteins at pharmaceutical levels.

Other efforts use purified translation systems to construct minimalcells using a bottom-up approach. Multiple groups have demonstrated theability to activate protein synthesis inside of an artificial liposometo more closely mimic native conditions (Murtas et al., 2007, Biochem.Biophys. Res. Commun. 363:12-17). Although such systems hold promise forlarge-scale protein screening and analysis, they are not adequate formanufacturing protein biopharmaceuticals at pharmaceutically-relevantlevels.

Examples of related art include: U.S. Pat. No. 7,338,789 describingmethods for in vitro synthesis of biological macromolecules underconditions and in a reaction composition wherein oxidativephosphorylation is activated and protein folding is improved; U.S. Pat.No. 8,357,529 describing methods for the enhanced in vitro synthesis ofbiological molecules; U.S. Pat. No. 6,780,607 describing methods ofproduction of completely post-translationally modified proteins bycombination of cell-free protein synthesis and cell-free co- andpost-translational modification in reticulocytes lysates, as well asmethods of supplementing those lysates with endoplasmic reticulum, Golgiand plasma membranes obtained from a Chinese Hamster Ovary (CHO) cells;and U.S. Pat. No. 8,034,581 disclosing a method for insect cell-freetranslation and post-translation glycosylation of target proteins, aswell as conditions for cell rupture and preparation of lysates carryingtranslation factors and factors with glycosylation activity.

SUMMARY

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification and a study of the drawings.

In one aspect, a portable, modular fluidic platform and method for rapidand flexible end-to-end cell-free expression, purification andformulation of recombinant protein biologics at the μg-mg scale isprovided. The platform comprises (a) a front end module that selects oneor more reaction reagents (which may include DNA, cDNA or mRNA template,a particular type of lysate, and one or more appropriate buffers) andloads them into a reactor module, (b) a reactor module for proteinproduction under continuous exchange cell-free (CECF) protein synthesisformat using prokaryotic or eukaryotic cell lysates; (c) a tandemprotein purification and concentration module for separation of thetarget protein(s) from the lysate mixture; and (d) a module for proteinpolishing and formulation. The platform incorporates components formixing, a reaction chamber, and a mechanism for moving reaction materialthrough purification modules, and allows addition and removal of wastematerials as required. The portable, modular fluidic platform and methodprovided herein allow the production, purification and formulation ofthe protein to be completed within a period of 24 hours. The process isflexible, scalable and amenable to automation for rapid production atthe point of need of proteins with significant pharmaceutical, medical,or biotechnological value.

In some embodiments, the platform further comprises one or more of thefollowing options: a control system that allows purification of multipleproteins on the same platform via switching among differentchromatography columns depending on target protein modalities; samplingports that allow for removal of small sample aliquots for qualitycontrol and assessment of protein synthesis quality, levels and/orpurity; syringe pumps with integrated rotor valves for direction offluidics between various protein synthesis and purification/formulationmodules allowing addition of appropriate solvents and removal of wastestream materials as required; swappable modules; and/or a fluidicallyintegrated protein synthesis and purification module into aself-contained disposable, single-use fluidic cartridge that eliminatesany possibility of cross-contamination between runs.

In one aspect, a cross-kingdom heterologous, cell-free translationsystem and method are provided for in vitro synthesis of protein targetscarrying mammalian PTMs. The system comprises (a) a DNA or mRNA templateencoding a target protein carrying a signal peptide for mammalianER-targeting, (b) a yeast or bacterial cell-free translation lysate fortranslation of exogenously added DNA and/or mRNA; and (c) a mammalianpost-translational modification extract comprising signal recognitionparticles (SRPs) and ER/Golgi microsomes for translocation andprocessing of the nascent protein.

Additional embodiments of the present platform and method, and the like,will be apparent from the following description, drawings, examples, andclaims. As can be appreciated from the foregoing and followingdescription, each and every feature described herein, and each and everycombination of two or more of such features, is included within thescope of the present disclosure provided that the features included insuch a combination are not mutually inconsistent. In addition, anyfeature or combination of features may be specifically excluded from anyembodiment of the present disclosure. Additional aspects and advantagesof the present disclosure are set forth in the following description andclaims, particularly when considered in conjunction with theaccompanying examples and drawings.

Specifically, this disclosure is directed toward a portable fluidicplatform for rapid and flexible end-to-end production of recombinantprotein biologics. The platform consists of a bioreactor system hostingstable and robust cell-free translation systems that is fluidicallyintegrated with modular protein separation functionalities (e.g., sizeexclusion, ion exchange or affinity chromatography systems) forpurification of the cell-free expressed product and which areconfigurable for process-specific isolation of different proteins, aswell as for formulation. The bioreactor utilizes lysates from engineeredeukaryotic (e.g., yeast) or prokaryotic (e.g., bacterial) strains thatcontain factors for protein folding and posttranslational modifications.Combination of various purification modules on the same fluidic platformallows flexibility of re-routing for purification of different proteinsdepending on specific target requirements. Protein synthesis andpurification modules are integrated into self-contained disposablefluidic cartridge that eliminates cross-contamination between runs.Analytical modules (i.e., capillary electrophoresis, E-PAGE) areintegrated into critical check-points to sample and monitor the process(e.g., assess protein purity levels and determine potential synthesis-or degradation-related impurities) and facilitate decision making duringthe production course and prior to product release. Protein yield isdetermined in-line using label-free methods on a small fraction of thesample. In vitro assays (specific to each target protein) are used in-or off-line to assess activity of the produced protein biologic. Theplatform allows for flexible production of protein biologics within 24hours (from DNA to purified product).

The platform can be used in cost-effective production of limited dosagesof biologics against rare medical conditions (orphan drugs). In anotherapplication, the platform can be used to enable fast (within 24 hours)synthesis and testing of proteins at small doses toward identificationof lead molecules during research and development efforts. In anotheruse, the platform can be used to provide on-demand ability to generatemedications in areas where those are not available due to refrigerationrequirements. In another application, the platform can be used inbattlefield medicine to increase medical capabilities of far forwardproviders and enable specific threat response.

The platform described herein exploits the unique properties of CFPSsystems for rapid protein production from DNA templates that do notrequire cell cultures or insertion of DNA sequences into cells. Theexpression system can be manipulated and optimized, thus avoiding theunpredictability of living systems. The bioreactor utilizes low costlysates from engineered eukaryotic (e.g. yeast) and prokaryotic (e.g.bacterial) strains that contain factors for protein folding andposttranslational modifications. The freedom of design afforded bycell-free production enables eukaryotic and prokaryotic lysates to beused interchangeably for expression of difficult protein targets. Thisflexibility yields a general expression platform that scales from two tohundreds of protein biologics, because different proteins can beexpressed preferentially in either eukaryotic or prokaryotic systems bysimply changing template DNA input. The platform concept also combinesan in-line flexible configuration of posttranslational, purification(e.g. ion exchange, size exclusion and affinity chromatography),formulation and characterization modules for efficient production ofactive protein biologic depending on target requirements. Integratedsystem allows flexibility of fluidics rerouting for purification ofdifferent target proteins on same platform. Such modularity also offersscalability through parallelization.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a schematic diagram of the fluidic process forcell-free production of protein biologics;

FIGS. 2A through 2F illustrate fluidic processes used in preparation,concentration, purification and formulation of protein biologics;

FIG. 3 illustrates a fluidic card for production of protein biologics;

FIGS. 4A and 4B illustrate protein synthesis reactors for continuousexchange protein synthesis;

FIG. 5 illustrates protein yield data using a fluidic reactor system;

FIG. 6 illustrates expression of human Erythropoietin from an expressionplasmid using a yeast cell-free protein synthesis system;

FIG. 7 illustrates in vitro production of human Erythropoietin in ayeast cell-free protein synthesis system combined with the mammalianposttranslational apparatus;

FIGS. 8A and 8B illustrate schematic representations of (8A) anaffinity-tag approach for purification of a protein product from crudelysates, and (8B) a purification scheme for separation of human GM-CSFfrom a bacterial cell-free protein synthesis system;

FIG. 9A through 9D illustrates purification data of human Erythropoietinexpressed in a yeast cell-free protein synthesis system;

FIG. 10 illustrates bioactivity data of human Erythropoietin proteinexpressed in a yeast cell-free protein synthesis system and purifiedusing an affinity chromatography system;

FIG. 11 illustrates expression of human Granulocyte-MacrophageColony-Stimulating Factor (GM-CSF) from an expression plasmid using abacterial cell-free protein synthesis system;

FIG. 12 illustrates purification of GM-CSF expressed in a bacterialcell-free protein synthesis system;

FIG. 13 illustrates activity data of GM-CSF protein produced in abacterial cell-free protein synthesis system and purified using anon-tag approach;

FIG. 14 illustrates expression of GM-CSF carrying an affinity tag in abacterial cell-free protein synthesis system;

FIG. 15 illustrates activity data of GM-CSF expressed in a bacterialcell-free protein synthesis system and purified using an affinitychromatography approach;

FIG. 16 illustrates activity data of human Erythropoietin expressed in abacterial cell-free protein synthesis system and purified using anaffinity chromatography approach;

FIG. 17 illustrates in vitro production of human erythropoietin in abacterial cell-free protein synthesis system combined with the mammalianposttranslational apparatus;

FIG. 18: illustrates Trypsin and Glu-C-digestion peptide products ofrhEPO expressed in the yeast CFPS system;

FIG. 19: shows the LC-MS/MS analysis of trypsin-digested FLAG™-EPOexpressed in the yeast CFPS system;

FIG. 20: shows the LC-MS/MS analysis of Glu-C-digested de-FLAG-EPO uponenterokinase treatment;

FIG. 21: illustrates Trypsin and Glu-C-digestion peptide products ofGM-CSF expressed in the yeast CFPS system;

FIG. 22: shows the LC-MS/MS analysis of trypsin-digested GM-CSFexpressed in the bacterial CFPS system; and

FIG. 23: shows the LC-MS/MS analysis of Glu-C-digested GM-CSF expressedin the bacterial CFPS system.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is the amino acid sequence of a triple FLAG™-tag.

SEQ ID NO: 2 is the amino acid sequence of GM-CSF (including signalpeptide).

SEQ ID NO: 3 is the amino acid sequence of GM-CSF (without signalpeptide).

SEQ ID NO: 4 is the amino acid sequence of FLAG™-EPO.

SEQ ID NO: 5 is the amino acid sequence of EPO (after FLAG™ tagcleavage).

SEQ ID NO: 6 is the forward PCR primer used for generating a templatefor the expression of rhEPO in yeast.

SEQ ID NO: 7 is the reverse PCR primer used to generate a template forthe expression of rhEPO in yeast.

DETAILED DESCRIPTION

Various aspects now will be described more fully hereinafter. Suchaspects may, however, be embodied in many different forms and should notbe construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey its scope to those skilled in theart.

I. Definitions

As used in this specification and appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a protein” includesa single protein as well as two or more of the same or differentproteins, reference to an “a cell” includes a single cell as well as twoor more of the same or different cells, and the like.

As used herein, “protein biologics” refer to protein- or peptide-basedproducts produced by recombinant DNA technology and can include, forexample, protein therapeutics, tissue (including blood) protein factors(e.g., factor VIII, thrombolytic agents, hormones, growth factors,interferons and enzymes), vaccines, monoclonal antibodies, and receptormolecules.

As used herein, a “protein of interest” or “POI” is any protein, orfunctional fragment (such as a protein domain) or derivative thereof,that one skilled in the art wishes to study.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably and without distinction to refer to a compound made upof a chain of amino acid residues linked by peptide bonds. Unlessotherwise indicated, the sequence for peptides is given in the orderfrom the “N” (or amino) terminus to the “C” (or carboxyl) terminus. Itis understood that polypeptides include a contiguous sequence of aminoacid residues.

As used herein, the terms “domain” and “region” are used interchangeablyherein and refer to a contiguous sequence of amino acids within aprotein, typically characterized by being either conserved or variableand having a defined function, such as ligand binding, conferringstability or instability, enzymatic function, etc.

As used herein, “conservative amino acid substitutions” aresubstitutions that do not result in a significant change in the activityor tertiary structure of a selected polypeptide or protein. Suchsubstitutions typically involve replacing a selected amino acid residuewith a different residue having similar physico-chemical properties. Forexample, substitution of Glu for Asp is considered a conservativesubstitution since both are similarly-sized negatively-charged aminoacids. Groupings of amino acids by physico-chemical properties are knownto those of skill in the art.

As used herein, a “variant” protein is a protein having an amino acidsequence that may or may not occur in nature, as exemplified bysequences in GenBank. As used herein, a “mutant” is a mutated proteinthat may occur in nature, or may be designed or engineered such that itsproperties (e.g., stability) or functions (e.g., ligand binding) arealtered.

A peptide or peptide fragment is “derived from” a parent peptide orpolypeptide if it has an amino acid sequence that is homologous, but notidentical, to the parent peptide or polypeptide. A peptide may be orrepresent a fragment of the parent protein or polypeptide.

Two amino acid sequences or two nucleotide sequences are considered“homologous” if they have an alignment score of >5 (in standarddeviation units) using the program ALIGN with the mutation gap matrixand a gap penalty of 6 or greater (Dayhoff, M. O., in Atlas of ProteinSequence and Structure (1972) Vol. 5, National Biomedical ResearchFoundation, pp. 101-110, and Supplement 2 to this volume, pp. 1-10.) Thetwo sequences (or parts thereof) are more preferably homologous if theiramino acids are greater than or equal to 50%, 70%, 80%, 90%, 95%, oreven 98% identical when optimally aligned using the ALIGN programmentioned above.

A “small molecule ligand” is a discrete small-molecule, well known inthe pharmaceutical and material sciences, which is to be distinguishedfrom, e.g., a polypeptide or nucleic acid polymer consisting ofmonomeric subunits. Small molecule ligands may be naturally-occurring orsynthetic as exemplified by pharmaceutical products, laboratoryreagents, and the like.

“Modulate” intends a lessening, an increase, or some other measurablechange in the stability or biological function of a protein.

As used herein, “preferentially binds” means to bind with greaterefficiency to a subject molecule (such as a receptor or other bindingpartner) than to another molecule. The difference in binding efficiencymay be 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1,000-fold, 10,000fold, or more.

As is known in the related art, a “breadboard” is a reusableconstruction base.

Where a range of values is provided, it is intended that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the disclosure. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is alsoencompassed. The upper and lower limits of the smaller ranges mayindependently be included or excluded in the range, and each range whereeither, neither or both limits are included in the smaller ranges isalso encompassed within the embodiments, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the embodiments. For example, if a range of1 μl to 8 μl is stated, it is intended that 2 μl, 3 μl, 4 μl, 5 μl, 6μl, and 7 μl are also explicitly disclosed, as well as a range of 2 μlto 7 μl, as is the range of values greater than or equal to 1 μl and therange of values less than or equal to 8 μl.

In some embodiments, a portable, modular platform for cell-freeproduction, purification and formulation of a protein is provided. Insome embodiments, the platform comprises a loading module for loadingone or more reaction reagents to a reactor module; a reactor modulecomprising at least one of prokaryotic and eukaryotic cell lysatemixtures, wherein said reactor module receives the one or more reactionagents from the loading module, and wherein said reactor modulefacilitates production of a target protein via a process selected from:(i) continuous exchange cell-free (CECF) or (ii) batch proteinsynthesis; a chromatography module for separating the target proteinfrom the lysate mixtures; and a finishing module for polishing andformulation of the target protein; wherein the production, separationand formulation of the target protein are completed within a period of24 hours.

In some embodiments, the production, separation and formulation of thetarget protein are completed within a period of 12 hours. In someembodiments, the production, separation and formulation of the targetprotein are completed within a period of 10 hours.

In some embodiments, the reactor module comprises a 2 mlpolydimethylsiloxane (PDMS) chamber. In some embodiments, the reactormodule holds a reaction volume between 0.1 and 5 ml. In someembodiments, the reactor module consists of a 2 ml PDMS chamber dividedby a 3.5 kDa dialysis membrane into reaction and feeding chambersholding 1 mL each.

In some embodiments, the loading module includes a feeder solution and asample solution (also known herein as a reaction solution). In someembodiments, the finishing module includes formulation via a sizeexclusion chromatography matrix. In some embodiments, the chromatographymodule for separating the target protein employs at least one of (a)FLAG™-tag-based affinity chromatography, (b) protease affinitychromatography, and (c) size exclusion chromatography steps, or acombination thereof. In some embodiments, the chromatography module forseparating the target protein employs at least one of (a) ion-exchangechromatography, (b) protein concentration, and (c) size exclusionchromatography steps, or a combination thereof.

In some embodiments, the lysates comprise a eukaryotic cell-free proteinsynthesis system. In some embodiments, the lysates comprise aprokaryotic cell-free protein synthesis system.

In some embodiments, the platform comprises cell-free co- andpost-translational modification using mammalian (including CHO cell andreticulocyte) lysates.

In some embodiments, the platform further comprises a control system forfacilitating purification of different proteins on the same platform viaswitching among various chromatography matrices. In some embodiments,the platform further comprises one or more sampling ports for removingsample aliquots for assessment of protein synthesis and formulationprocesses. In some embodiments, the platform further comprises one ormore syringe pumps with integrated rotor valves for directing fluidicsbetween one or more modules and allowing addition or removal of asolution. In some embodiments, the platform further comprises afluidically integrated protein synthesis and purification module into aself-contained disposable fluidic cartridge.

In some embodiments, a heterologous cross-kingdom cell-free translationsystem for in vitro synthesis of protein targets carrying mammalian PTMsis provided. In some embodiments, the cell-free translation systemcomprises one or more of a yeast or bacterial cell-free lysate fortranslation of exogenously added polynucleotide selected from the groupconsisting of DNA and mRNA; one or more of an exogenous DNA or mRNAtemplate encoding a target protein carrying a signal peptide formammalian ER-targeting; and a mammalian post-translational modification(PTM) extract comprising signal recognition particles (SRPs), ER andGolgi microsomes, or a combination thereof, for translocation andprocessing of the target protein(s).

In some embodiments, a method of producing a small amount of a proteinbiologic formulation at pharmaceutical dosage levels at a point of needusing the platform described above is provided, wherein steps ofproduction, separation and formulation of the protein biologicformulation are completed within a period of 24 hours.

In some embodiments, between about 40 μg/ml and about 8 mg/ml protein isproduced. In some embodiments, between about 40 μg/ml and about 2 mg/mlprotein is produced. In some embodiments, between about 500 μg/ml andabout 2 mg/ml protein is produced.

As used herein, a reaction solution is a mixture containing the lysate,polynucleotide template and enzymes and reagents required for DNAtranscription and protein synthesis. As used herein, a feeder solutionhas the same composition as the reaction solution except for the absenceof lysate, polynucleotide template and DNA transcription enzyme (forexample, T7 polymerase).

This disclosure is directed to a portable fluidic platform for rapid andadaptive cell-free synthesis of multiple protein biologics (FIGS. 1,2A-2F and 3). In this platform, translationally active lysates—preparedfrom eukaryotic or prokaryotic cells—are programmed for coupledtranscription/translation of an engineered DNA template encodingspecific protein targets.

Cell-free protein production offers several advantages overconventional, in vivo, protein expression methods: for example in acell-free system, most of the subcellular metabolic resources can bedirected toward the production of a protein, and the in vitro synthesisenvironment is more easily controlled absent a cell wall and certainmembranous components. Because cell growth or viability are no longerconsiderations in cell-free protein production systems, conditions suchas redox potential, pH, or ionic strength can be altered with greaterflexibility than in the case of in vivo protein production. In vitrotranslation is also recognized for its ability to incorporate unnaturaland isotope-labeled amino acids as well as its capability to produceproteins that are unstable, insoluble, or cytotoxic in vivo.Furthermore, components such as tRNA levels can be changed to reflectthe codon usage of genes being expressed. Direct recovery of purified,properly folded protein products is readily achieved, and cell-freesystems have allowed production of proteins in the mg/ml range.

Regarding a prokaryotic cell-free protein synthesis platform, bacterialEscherichia coli provides a suitable source organism since: a) it is anFDA approved microorganism for protein production, b) it allows forinexpensive fermentation in large quantities using low-cost media, c) ithas the lowest CFPS reaction cost among other lysates, d) Generateshigher protein yields than other lysates, and e) engineered systemsenable posttranslational modifications, including S—S and N-linkedglycosylation.

Regarding a eukaryotic cell-free protein synthesis platform, yeastSaccharomyces cerevisiae can be used as a source organism based onseveral attractive features, such as: a) it is an FDA-approvedmicroorganism for protein production, b) it provides inexpensive andscalable methods for cell cultivation and lysate production, c) itsgenome is well characterized genome and there are advanced techniquesfor its genetic manipulation, and d) it is suitable for synthesis ofcomplex proteins and glycosylation patterns.

The platform disclosed herein allows using both E. coli and S.cerevisiae CFPS systems to capitalize on the advantages of each platformfor expression and manufacture of different protein biologics withvarying requirements for expression, PTM, folding kinetics, and scalerequirements, such as quantity/therapeutic dose. The flexibility of theplatform also allows use of other CFPS systems originating fromeukaryotic (e.g., mammalian, plant, insect) or prokaryotic (e.g.,archaea or bacterial) organisms. Using methods that are amenable to afully automated approach, the presently described protein biologicsmanufacturing platform exemplifies the expression, purification andformulation of two pharmaceutically relevant biologics, recombinanthuman Erythropoietin (rhEPO) and recombinant humanGranulocyte-Macrophage Colony-Stimulating Factor (rhGM-CSF).

Several approaches can be used to maximize protein expression in CFPSsystems, such as messenger RNA (mRNA) engineering (e.g., use of strongpromoters and/or internal ribosome entry sites), genetic engineering ofthe source strain(s), and biochemical optimization of the cell-freesystem(s). Supplementation of these in vitro systems with Endoplasmicreticulum (ER) and Golgi membranes from mammalian cells (e.g. ChineseHamster Ovary (CHO) cells) allows for synthesis of therapeutically ordiagnostically relevant amounts of human biologics carrying appropriateposttranslational modifications for correct folding, high activity andappropriate pharmacological properties. Furthermore, creation of anoxidizing environment that favors disulfide bond formation in targetproteins and supplementation of the in vitro system with specificfoldases and chaperone molecules allows for correct protein folding andenables synthesis of soluble and active target proteins.

Protein synthesis takes place in bioreactors under batch or CECFformats. An exemplary batch reaction for bacterial CFPS protocols can beperformed according to conditions described in Goerke and Swartz (2008,Biotechnol. Bioeng. 99:351-367); Iskakova et al. (2006, Nucleic AcidsRes. 34, e135); or Brandi et al. (2008, Methods Mol Med. 142:87-105).

An exemplary continuous exchange reaction for bacterial CFPS protocolscan be performed according to conditions described in Shirokov et al.(2007, Methods Mol. Biol. 375:19-55).

An exemplary batch reaction for yeast CFPS protocols can be performed asdescribed in Brandi et al. (2008, Methods Mol Med. 142:87-105) orHodgman and Jewett (2013, Biotechnol. Bioeng. 110, (10):2643-2654).

An exemplary continuous exchange reaction for yeast CFPS protocols canbe performed according to Schoborg et al. (2014, Biotechnol. J.9:630-640).

In the CECF format, bioreactors consist of a dialysis membranesandwiched between the reaction and feeding chambers, which are made ofbiocompatible materials. This format allows for replenishment ofsubstrates and removal of low molecular-weight products from thereaction by diffusional exchange across the membrane. CECF systems havebeen utilized for generation of long-lived, highly-productive in vitroprotein synthesis systems using both prokaryotic and eukaryotic lysates.

Various methods can be employed for purification of cell-free producedproteins in the fluidic platform. These methods include use of variouschromatographic materials, such as affinity, ion exchange, and sizeexclusion matrixes. In the case of glycosylated proteins (e.g.,erythropoetin), CFPS systems can also be subjected tocarbohydrate-affinity chromatography (e.g., lectin-affinity) forspecific separation of glycoproteins from the complex lysate accordingto the structure of their glycan moieties. Quantitative techniques(e.g., colorimetric immunochemical assays or interferometry) can enabledetermination of protein recovery. Chromatographic techniques candetermine purity levels, whereas the mass of the protein andglycosylation sites can be identified by mass spectroscopy. The qualityof the final protein sample can be assessed using a series of commonprotocols in biopharmaceutical manufacturing (Chirino and Mire-Sluis,2004, Nat. Biotechnol. 22:1383-1391).

Integration of the bioreactor with modular functionalities for flexibleprotein purification, formulation, quantification, activity evaluationand characterization of critical impurities, creates a single, portableplatform capable of generating biologically-derived proteins within 24hours. The system can be designed around self-contained disposableprotein synthesis and separation cartridges for elimination ofcross-contamination between runs. The cartridges can be temperaturestable at ambient temperatures, and individually packaged. In addition,the system can potentially use lyophilized reagents that are hydratedautomatically within the device, enabling long-term reagent storage andfield use.

Candidate protein biologics produced in the aforementioned platform mayinclude virtually any protein FDA-approved therapeutic (Leader et al.,2008, Nat. Rev. Drug. Discov. 7:21-39). One non-limiting example isrecombinant human Epoetin alpha, a 166 amino acid glycoprotein (MW˜34KDa) produced by recombinant DNA technology, typically in CHO cells.Epoetin alpha is the recombinant version of endogenous humanerythropoietin (EPO; or “rhEPO”), a protein that regulates theproduction of erythrocytes. Intravenous administration of Epoetin alphais used to treat anemia caused by renal failure; in addition, it hasbeen shown to have tissue protective, neuroprotective andcardioprotective activity upon injury (Mocini et al., 2007, Curr. Med.Chem. 14:2278-2287).

Another non-limiting example is recombinant human Granulocyte-MacrophageColony-Stimulating Factor (rhGM-CSF), a 143 amino acids (MW˜16 kDa)multi-disulfide-bonded cytokine that functions as a white blood cellgrowth factor and is part of the immune/inflammatory cascade (Metcalf,1986, Blood. 67:257-67). This factor stimulates stem cells to producegranulocytes and monocytes which mature into macrophages and dendriticcells, a process that is crucial for fighting infection.

Another non-limiting example is Hepatitis B Vaccine (purified surfaceantigen HBsAg) that is currently produced from yeast cultures.Administration of this vaccine reduces incidence of HBV infection and isrecommended for military personnel traveling to areas where HBV isendemic.

This disclosure provides cell-free protein synthesis platforms andsystems (prokaryotic and eukaryotic) for flexible, scalable, automatableand on-demand expression of a protein biologic at therapeutic doseamounts, wherein the production, purification and characterizationprocess can be completed within 24 hrs. In the platform disclosedherein, the CECF protein synthesis reaction is completed within 12 hoursof incubation followed by an additional ˜12 hours during which theprotein is purified and formulated. In some embodiments, the proteinsynthesis step can be completed within shorter duration periods (e.g.,approximately 2 hours) depending on protein yield requirements. In someembodiments, the purification and formulation process can be completedwithin shorter duration periods (e.g., approximately 4-8 hours)depending on the protocol requirements for the specific protein target.In some embodiments, the production, purification/separation andformulation processes are completed within a period of 12 hours. In someembodiments, the production, purification/separation and formulationprocesses are completed within a period of 10 hours.

In the platforms disclosed herein, modular fluidic cartridges wereengineered/fabricated and tested for protein synthesis and purification(size exclusion, ion exchange and affinity chromatography). Fluidicreactors for protein synthesis under continuous exchange format werefabricated and tested using 1 and 5 ml reaction volumes. The reactorswere implemented with fluidic connectors to allow recirculating flow andfluidic integration with upstream reagents mixing and downstreampurification modules. These reactors successfully supported cell freeprotein synthesis under continuous exchange format, resulting in 8-foldhigher yields than batch format, and ˜75% yields compared to COTS(Commercial-Off-the-Shelf) CECF reactors, and have been used inend-to-end experiments.

The “end-to-end produced” EPO and GM-CSF protein products weredemonstrated to be bioactive, i.e., the EPO and GM-CSF proteins producedin the bioreactor and purified using the chromatographic modulesprovided herein had similar biological activities to those of commercialcontrol proteins.

Nascent polypeptides synthesized in vitro using the platform/methoddisclosed herein are soluble and accurately processed enzymatically,resulting in mature proteins with sequences identical to those ofpharmaceutical products (Sargramostim™ for GM-CSF and Epogen™ for EPO).Liquid Chromatography-MS/MS confirmed that the N-terminus amino acidsequences of both target proteins are identical to those of the actualpharmaceuticals (i.e., they carry the natural amino acid sequence and donot start with methionine as it is often the case with in vitroexpression systems as a means of increasing protein yields).

The nascent polypeptides also bore PTMs: the bacterial cell-free systemgenerated disulfide bonds in the GM-CSF produced, and a fraction of theEPO protein was glycosylated in a reconstituted heterologous systemconsisting of mammalian ER and Golgi microsomes mixed with either yeastor bacterial lysates.

In some embodiments, it is advantageous to use DNA templates (e.g.,plasmids or PCR templates) that carry sequences that enhance proteinproduction in the bacterial CFPS system. Such templates have beensuccessfully used in the platform disclosed herein to accomplish agreater than 50-fold enhancement in protein expression as compared tothe previous state of the art methods/systems. For example, humanrecombinant GM-CSF bearing the sequence of the pharmaceutical productSargramostim™ was produced at >2 mg per ml of reaction. In addition, bysimply changing DNA template, the system allowed production of humanrecombinant EPO (Epogen™) at >8 mg per ml of reaction.

In some embodiments, it is advantageous to use DNA templates (e.g.,plasmids or PCR templates) that carry sequences that enhance proteinproduction in the yeast CFPS system. Such templates have beensuccessfully used in the platform disclosed herein to accomplish agreater than 20-fold enhancement in protein expression as compared tothe previous state of the art methods/systems. For example, humanrecombinant EPO (Epogen™) was produced at ˜40 μg per ml of reaction.

A modular strategy for fluidic purification of proteins from prokaryoticand eukaryotic cell-free systems within 12 hrs was developed.Purification yields for GM-CSF and EPO were ˜30% and 50%, respectively,allowing generation of single dose amounts of protein products fromsmall cell free reaction volumes (1-5 ml).

Rapid Cell-Free Expression and Purification of Protein Biologics

The process provided herein was developed for rapid production ofprotein biologics at low doses. The process consists of combinedtranscription/translation of a DNA template encoding for the targetprotein under CECF or batch formats followed by a series of purificationsteps for separation of the protein biologic from the components of thecell-free system. The modularity of this process allows flexibility inusing prokaryotic or eukaryotic lysates as well as combination ofdifferent protein purification workflows for production of variousproteins depending on specific target modalities. This process was usedto demonstrate in vitro synthesis and purification of active rhEPO andrhGM-CSF, expressed in S. cerevisiae and E. coli lysates, respectively,in less than 24 hours. These proteins contained the coding sequences forthe FDA-approved protein products, Epoetin alfa, a recombinant versionof endogenous hEPO, and Sargramostim™, a recombinant version ofendogenous hGM-CSF.

Development of a Flexible Process for Production of Protein Biologics

The developed process exploits the unique properties of CFPS systems forrapid protein production from DNA templates that do not require cellcultures or insertion of DNA sequences into cells. Both S. cerevisiaeand E. coli CFPS systems were employed to capitalize on the advantagesof each platform for expression of different protein biologics withvarying requirements. E. coli provides a suitable source organism sinceit is an FDA approved microorganism for protein production, it allowsfor inexpensive fermentation in large quantities using low-cost media,it has the lowest CFPS reaction cost among other lysates, and itgenerates the highest protein yields of all other lysates (Carlson etal., 2012, Biotechnol. Adv. 30:1185-1194). S. cerevisiae was used as asource organism because, similarly to E. coli, it is an FDA-approvedmicroorganism, it provides scalable methods for cell cultivation andlysate production, it is suitable for synthesis of complex proteins, itsgenome is well characterized and there are advanced techniques for itsgenetic manipulation. The process described herein affords flexibilityand enables S. cerevisiae and E. coli CFPS systems to be usedinterchangeably for expression of different therapeutics with varyingbiochemical and pharmacological requirements. As described herein, rhEPOwas expressed in the yeast CFPS system whereas rhGM-CSF was produced inthe bacterial CFPS system. Parallel experiments showed production ofrhEPO in the bacterial CFPS system at yields that were significantlyhigher than those obtained in the yeast system and similar to those ofrhGM-CSF produced in the bacterial CFPS system, and with activities thatwere comparable to that of a commercially available control EPO protein(results to be described elsewhere). The flexibility of the describedprocess also opens the possibility to use lysates from any organism forsuch purpose. In addition, reaction volumes in these systems can bescaled up through the use of larger reactors and/or parallelization toallow production of higher doses of protein biologics as needed.

The protein purification principles applied in the present work werefocused on utilizing well-characterized resins and establishedapproaches widely used for purification of protein biologics. Themodularity of the purification processes, in combination with theflexibility of fluidics routing, can make possible the purification ofdifferent proteins on the same platform starting from DNA expressed intheir respective CFPS system and continuing through the particularpurification scheme optimized for each protein. In addition, completeautomation is feasible by putting syringe drives and valves undercomputer control and the development of robust protocols thatreduce/eliminate in-process decisional matrices.

Potential Applications of a Fluidic Process for Rapid Production ofProtein Biologics

Such a fluidic platform has important applications for the production ofprotein biologics. Proteins produced in the aforementioned platform mayinclude, for example, FDA-approved protein therapeutics (Leader et al.,2008, Nat. Rev. Drug. Discov. 7:21-39). The system can also be used forrapid, cost-effective, production of limited dosages of biologicsagainst rare medical conditions (orphan drugs), antibody vaccines, orprotein-based medical countermeasures. Alternatively, it can be used toenable rapid synthesis and testing of proteins at small doses for drugscreening or structure-function analysis during research and developmentefforts. Furthermore, it can be used for generating medications in areaswhere those are not available or in military medicine to increasemedical capabilities of far forward providers and/or enable specificthreat response.

The present disclosure enables production of pharmaceutical-gradeproteins by a fully automated portable platform, a capability that couldpotentially allow reduction of pharmaceutical manufacture to anintegrated fluidic process with a single dose lot size. Such a systemcan produce analytical and statistical data demonstrating that thedesigned platform can reproducibly deliver a product to an establishedspecification. The process can be optimized by using a Quality-by-Designapproach that incorporates appropriate in-process check points andcritical quality assurance assays in the final implementation of themanufacturing process to ensure that the limited lot size product meetsthose specifications. The present disclosure shows that such a platformis feasible for rapid production of low doses of protein biologics atthe point-of-need. In addition, the platform is scalable and amenable tointegration with fluidic analytical modules towards the generation of afully automated system for production of proteins with significantpharmaceutical, medical, or biotechnological value.

The following examples are illustrative in nature and are in no wayintended to be limiting.

Example 1 DNA Constructs

A plasmid encoding the protein sequence of rhEPO (Epoetin alfa; DrugBankAccession Number DB00016) was generated containing the 5′-UTR fragmentof the Omega sequence from the tobacco mosaic virus (Gan and Jewett,2014, Biotechnol. J. 9:641-651) and used to produce polymerase chainreaction (PCR) DNA templates for combined transcription/translation inthe yeast CFPS system. (see FIG. 6).

A plasmid encoding rhGM-CSF (Sargramostim™; DrugBank Accession NumberDB00020) was generated from the pY71 backbone (Bundy and Swartz, 2010,Bioconjug. Chem. 21:255-263) for use as a circular DNA template for thebacterial combined transcription/translation CFPS system (see FIG. 11).

Both constructs contained a T7 promoter and signal peptide sequencelocated upstream of the mature rhEPO and rhGM-CSF coding regions; therhEPO construct also contained three copies of a FLAG™ epitope locatedbetween the signal peptide and the protein coding region for use in anaffinity chromatography-based approach for protein purification. Signalpeptide and gene sequences were codon modified based on a commercialproprietary method (RESCUE; Promosome, LLC, CA). The PCR templates forthe yeast CFPS reactions were generated from the aforementioned rhEPOplasmid using a forward primer with the sequence:5′-GTGATTCATTCTGCTAACCAG-3′ (hereinafter identified as SEQ ID NO: 6),and a reverse primer with the sequence: 5′-T₅₀GTTAGCAGCCGGATCTCAGT-3′(wherein “T₅₀” means that the first 50 nucleotide residues at the 5′ endof the polynucleotide are thymine; the 20 residues following the T₅₀ arepresented as SEQ ID NO: 7). PCR reactions were performed using Phusion®High-Fidelity DNA Polymerase (New England Biolabs, Inc., MA), 0.2 μM ofeach primer, and 0.1 ng/μl of template DNA. The PCR product was purifiedusing QIAquick™ PCR Purification Kit (Qiagen, MD), quantified using aNanoDrop™ 1000 (Thermo Fisher Scientific, MA), and its quality assessedby agarose gel electrophoresis.

Example 2 Protein Synthesis Reactor

A computer-aided design model of the protein synthesis reactor forhosting cell free protein synthesis under continuous exchange format isshown in FIG. 4. Dotted features are “stand-off” supports to maintainchannel volume by constricting dialysis membrane motion (FIG. 4A). FIG.4B is a photograph of a protein synthesis reactor, feeder and sampleloading modules. The arrow at 1 in FIG. 4B shows a protein synthesisreactor, consisting of a 2 ml polydimethylsiloxane (PDMS) chamberdivided into the reaction and feeding chambers (1 ml, each) by a 3.5 kDamolecular weight cut-off regenerated cellulose dialysis membrane(Spectra/Por 7 Dialysis Membrane, VWR, PA). In some embodiments, bothhalves of the reactor were implemented with 0.010-0.030 ID polyethylene(PE) tubing fluidic systems (Western Analytical Products, CA) to allowfor recirculating flow, fluidic integration of the reactor module withthe upstream feed and sample reagents modules, and for reaction materialrecovery. In some embodiments, molded PDMS chambers were produced from astereo-lithographic printed mold. The arrow at 2 in FIG. 4B shows aperistaltic pump and storage vial for feeder solution loading to thefeeding chamber of the reactor and recirculation. The arrow at 3 in FIG.4B shows a diaphragm and vial for reagents loading to the reactorchamber and sample recovery.

Example 3 Mass Spectrometry

For protein mass spectrometric analysis, protein samples were analyzedin 4-12% SDS-PAGE gels and stained with SimplyBlue SafeStain (LifeTechnologies Corporation, CA) according to the manufacturer'srecommendations. Gel bands migrating to the same molecular weight as thecomparative control were excised and mass spectrometry was performed atthe facilities of The Scripps Research Institute Center for MassSpectrometry (TSRI, CA). The gel bands were destained, reduced (10 mMDTT), alkylated (55 mM idoacetamide), and digested with trypsin or Glu-Covernight before being analyzed by nano-LC-MS/MS.

Example 4 Protein Activity Assays

The bioactivity of the purified recombinant human EPO and GM-CSF wasassessed using the TF-1 cells proliferation assay; this cell line isderived from bone marrow cells and exhibits growth dependency on bothEPO and GM-CSF proteins. (Kitamura et al., 1989, Blood 73:375-380).Briefly, TF-1 cells (ATCC # CRL 2003) were maintained in RPMI 1640 media(ATCC 30-2001) supplemented with Fetal Bovine Serum (10%),Penicillin/Streptomycin (100 U/ml) and rhGM-CSF (2 ng/ml), and grown at37° C. in the presence of 5% CO₂. For activity tests, cells were washedthree times in growth medium without GM-CSF, plated in 96-well cultureplates (2×10⁴-1×10⁵ cells/ml) in the presence of serial dilutions of thetest sample or control protein (final volume 100 μl/well), and incubatedat 37° C. for 48-72 hours. At the end of the incubation period, cellswere removed from 37° C. and allowed to equilibrate at room temperaturefor 30 min. An equal volume (100 μl) of room-temperature Cell Titer-Gloreagent (Promega Corporation, WI) was added to the cells followed byshaking of the plates at 900 rpm for two min. After incubation of theplates for 10 min at room temperature, luminescence was measured using aMithras LB 940 multimode microplate reader (Berthold Technologies,U.S.A. LLC, TN). Human EPO (CYT-201, ProSpec-Tany TechnoGene, Ltd, NJ)and GM-CSF (C003, Novoprotein Scientific, Inc., NJ) proteins were usedas controls. Data was analyzed and graphed using GraphPad Prism v6(GraphPad Software, Inc., CA).

Example 5 Development of a Fluidic Process for Rapid Production ofProtein Biologics

A breadboard system was developed for expression, purification andformulation of two separate therapeutic proteins via switching amongmultiple chromatography columns depending on target proteinrequirements. Schematic diagrams of the fluidic platform and a fluidiccard for production of protein biologics are depicted on FIGS. 1, 2A-2Fand 3.

As shown in FIG. 1, the fluidic process consists of: i) Reagents (feederand reaction/sample solutions) loading to the reactor system, ii)Combined transcription/translation step taking place in a reactor moduleunder continuous exchange or batch format, iii) Protein purificationsteps for separation of the target proteins from the lysate mixturesusing a variety of chromatography matrices depending on protein targetmodalities, and iv) Protein polishing (i.e., further purification,if/when necessary) and formulation step, typically through the use of asize exclusion chromatography matrix.

The process incorporates a reactor system for CFPS of biologic targetproteins using eukaryotic or prokaryotic lysates, modules for proteinpurification (affinity or ion exchange chromatographies) for isolationof expressed protein from the lysate mixture, and a module for proteinpolishing (i.e., further purification, if/when necessary) andformulation (size exclusion chromatography) and movement of reactionmaterial through purification modules, with addition and removal ofwaste materials as required. Temperature control devices can allowindependent temperature adjustment of all systems whereas qualitycontrol sample ports allow for removal of small aliquot samples fordownstream testing. System also allows flexibility of fluidic reroutingfor purification of different target proteins (e.g., EPO and GM-CSF) onthe same breadboard.

Reactions in batch systems typically plateau after a short period oftime (typically 2-3 hours). Potential shortage of critical components,such as amino acids and nucleotides, during protein synthesis, isresponsible for such reduction in the rate of protein production overtime (Shirokov et al., 2007, Methods Mol. Biol. 375:19-55). CECF proteinsynthesis systems allow replenishment of substrates and removal of lowmolecular-weight by-products by diffusional exchange across a membraneduring protein synthesis (Shirokov et al., 2007, Methods Mol. Biol.375:19-55). Accordingly, a fluidic reactor for protein synthesis undercontinuous exchange format was fabricated and tested (1 and 5 mlreaction volumes) was developed for supporting CECF protein synthesis.The reactor consists of a conventional regenerated cellulose dialysismembrane sandwiched between the reaction and feeding chambers, which aremade of biocompatible materials (PDMS). The reactor is implemented withfluidic connector tubing systems to allow recirculating flow in thefeeding chamber, fluidic integration with upstream reagent modules (forpossible mixing steps), and sample recovery for downstream purificationand processing (FIGS. 4A and 4B). The design allows for use of variousand alternative dialysis membrane types with different molecular weightcut-offs for optimum materials exchange as well as further volume scaleup through parallel connection of reactor modules depending on yieldrequirements. The design also allows for direct connection of the twochambers in the absence of a dialysis membrane for supporting CFPS undera batch format. Reactors successfully supported cell free proteinsynthesis generating ˜8-fold higher protein yields than in a batchformat and reaching ˜75% of the yields obtained in a COTS continuousexchange format reactor.

FIGS. 8A and 8B show schematic diagrams of the fluidic process forcell-free production of protein biologics. FIG. 8A presents a schematicrepresentation of the purification process for recovery of rhEPO from ayeast cell-free protein synthesis system. This method incorporatesFLAG™-tag-based affinity chromatography for protein recovery from thecell-free system with subsequent removal of the affinity tag forrecovery of the intended size protein target. FIG. 8B presents a processdiagram for purification of rhGM-CSF from a bacterial cell-free proteinsynthesis system via the use of anion-exchange chromatography incombination with size exclusion chromatography.

A modular protein purification strategy was developed for purificationof rhEPO from the yeast CFPS system (FIG. 8A). This strategy involvesincorporation of three copies of a FLAG™ hydrophilic peptide at theN-terminus of the target protein (see FIG. 6); the FLAG™-tagged proteintarget can then be purified using an immobilized monoclonal antibodymatrix under non-denaturing conditions and eluted by lowering the pH orby adding competing amounts of free FLAG™ peptide. An important featureof the FLAG™ tag is the inherent Enterokinase cleavage site located atthe C-terminus of the FLAG™ peptide sequence (Arnau et al., 2006,Protein Expr. Purif. 48:1-13; Young et al., 2012, Biotechnol. J.7:620-634). Enterokinase cleaves the FLAG™ epitope without requiring aspecific linker sequence and allows for the removal of the tag withoutleaving residual amino acids on the target protein (Arnau et al., 2006,Protein Expr. Purif. 48:1-13; Young et al., 2012, Biotechnol. J.7:620-634). Subsequently, the enzyme can be removed using anEnterokinase-affinity chromatography and the target protein can berecovered and further purified through a size exclusion chromatographystep (FIG. 8A). A modular strategy, similar to the method usedpreviously (Zawada et al., 2011, Biotechnol. Bioeng. 108:1570-1578), wasdeveloped for purification of GM-CSF from bacterial cell-free systemsusing a combination of ion exchange (DEAE Sepharose Fast Flow) and sizeexclusion (Sephacryl 5200) chromatography matrices (FIG. 8B).

Example 6 Yeast Cell-Free Protein Synthesis

The rhEPO constructs carrying signal peptide sequences upstream of thecoding region as well as a FLAG™ sequence between the signal peptide andthe coding region were subjected to coupled transcription/translation inthe yeast CECF protein synthesis system.

Saccharomyces cerevisiae strain S288c (Mortimer and Johnston, 1986,Genetics, 113:35-43) was used as the source strain for extractpreparation that was performed according to Hodgman and Jewett (2013,Biotechnol. Bioeng. 110:2643-2654) with the exception of growing theyeast cells on synthetic complete media (6.7 g/l Yeast Nitrogen Base(YNB), 20 g/l glucose, 50 mM potassium phosphate buffer, pH 5.5, and2.002 g/l Synthetic Complete Amino Acids Supplement (ForMedium™,Norfolk, United Kingdom). The yeast CECF combinedtranscription/translation protein synthesis reaction had the followingfinal composition: 22 mM HEPES, pH 7.4, 120 mM potassium glutamate, 5.5mM magnesium glutamate, 1.5 mM of each ATP, GTP, CTP, and UTP, 0.08 mMof each of 20 amino acids, 25 mM creatine phosphate, 1.7 mM DTT, 2 mMputrescine, 0.5 mM spermidine, 0.4 mM cAMP, 0.27 mg/ml creatinephosphokinase, 1.3 U/μl T7 polymerase (Thermo Fisher Scientific, MA),11% glycerol, 6.67 ng/μl PCR amplified DNA template, and 50% (v/v) yeastextract. The feeder solution (present in the feeder chamber of thereactor) had the same composition as the reaction mixture (present inthe reaction chamber of the reactor) except for the absence of T7Polymerase, DNA template, and extract and the presence of 115 mMmannitol. The reaction and the feeder solutions (1 and 10 ml,respectively) were loaded into the CECF reactor and incubated at roomtemperature for 6 hours with continuous recirculation of the feedersolution at a rate of 1 ml/min. To assess the solubility of theexpressed rhEPO in the CFPS system, upon incubation completion, reactionmixtures were centrifuged at 16,000×g for 10 min at 4° C., thesupernatant (soluble) fractions were isolated, the pelleted (insoluble)fractions were resuspended in the same volume of phosphate-bufferedsaline (PBS), and both samples were mixed with SDS loading buffer, heatdenatured and subjected to SDS-PAGE followed by Western blot analysis.Unless otherwise specified, reagents were obtained from Sigma-AldrichCorporation (St. Louis, Mo.). Initial testing of product yieldsindicated that under these conditions the yeast CFPS system had avariability of approximately 6% between different lysate lots, andvariability of less than 5% between replicate experiments using the samelysate lot on different days.

Western blot analysis of the EPO product showed that the expressedprotein was present mainly in the soluble fraction (FIG. 6). Nascentpolypeptides synthesized in vitro were also accurately processedenzymatically, generating mature proteins with sequences identical tothose of the pharmaceutical product (Epogen™). To this end, liquidchromatography-tandem mass spectrometry (LC-MS/MS) confirmed expectedtarget sequences for EPO produced in the yeast system (144 of 166 aminoacids were identified (86.7% coverage)). In addition, LC-MS/MS analysisconfirmed the presence of the correct N-terminal sequence in thecell-free human EPO protein product as the result of enzymatic affinitytag cleavage during the protein purification process.

Using the yeast CECF protein synthesis system, human EPO was produced at˜40 μg per ml of reaction. Reactions volumes in this system can bescaled up through the use of larger reactors and/or parallelization toallow production of greater amounts and/or higher therapeutic doses ofprotein biologics, as needed.

Example 7 Glycosylation of Human Erythropoietin in a Heterologous YeastCell-Free Protein Synthesis System

The cellular posttranslational machinery has been described previously(e.g., Walter et al., 1982, Philos. Trans. R. Soc. Lond. B. Biol. Sci.300:225-228; Walter et al., 1984, Cell, 38:5-8). This mechanism involvesassociation of the Signal Recognition Particles (SRPs) with signalpeptide (SP) sequences present in newly synthesized proteins destinedfor the Endoplasmic Reticulum (ER). SRP then carries theribosome/nascent peptide complex to the ER for binding to the SRPreceptor. The nascent protein is inserted into the translocon,translocated into the membrane of the ER, and the SP is cleaved by apeptidase. In the ER, the newly synthesized protein is associated withchaperones for proper folding followed by appropriate post-translationalmodifications (e.g., glycosylation).

CFPS systems capable of PTMs consist of three main parts: templateencoding a target protein carrying a signal peptide for ER-targeting; alysate capable of translating exogenously added cDNA/mRNA; and SRP andmembrane fractions of ER and Golgi microsomes for translocation andprocessing of the nascent protein. Past efforts have successfully usedhomologous yeast components (yeast lysate and yeast microsomes) for invitro synthesis and glycosylation of target proteins as well asheterologous systems made of reticulocyte lysates and microsomes fromcanine pancreas (e.g., Walter et al., 1982, Philos. Trans. R. Soc. Lond.B. Biol. Sci. 300:225-228; Walter and Blobel, 1983, Methods Enzymol.96:84-93; Walter et al., 1984, Cell, 38:5-8). However, successfulreconstitution of a heterologous cell-free translation system comprisingyeast lysate and mammalian microsomes has not yet been demonstrated.

To this end, a heterologous cross-kingdom reconstituted systemconsisting of a yeast cell-free translation system mixed with themammalian translocation machinery (ER/Golgi microsomes and SRPs isolatedfrom CHO cells) was developed for in vitro synthesis of protein targetscarrying mammalian PTMs. The isolation of mammalian membranes andenzymatic factors was performed using previously described methods(Walter and Blobel, 1983, Methods Enzymol. 96:84-93). The results usingthe aforementioned reconstituted system showed the generation of an EPOprotein product having a MW that was similar to that of the glycosylatedEPO control protein (FIG. 7).

Example 8 Purification of Human Erythropoietin from a Yeast Cell-FreeProtein Synthesis System

A modular generalized protein purification strategy was developed forprotein purification from a cell-free protein synthesis system (FIG.8A). This strategy involves incorporation of three FLAG™ tags at theN-terminus of the protein (DYKDDDDL; hereinafter identified as SEQ IDNO: 1 and shown in FIG. 6). FLAG™-tagged proteins can be purified usingan immobilized monoclonal antibody matrix under non-denaturingconditions and eluted by lowering the pH or adding competing amounts offree peptide. A unique aspect of FLAG™ is the inherent Enterokinasecleavage site located within the five C-terminal residues of the peptidesequence. Enterokinase cleaves the FLAG™ epitope without requiring aspecific linker sequence and allows for the complete removal of the tagwithout leaving residual amino acids on the target protein.Subsequently, the enzyme can be removed using an Enterokinase-affinitychromatography and the target protein can be recovered and furtherpurified through a size exclusion chromatography.

This strategy was used to purify human EPO from a yeast CECF proteinsynthesis system (FIGS. 9A through 9D). Purification of expressedFLAG′-tagged rhEPO protein from the yeast lysate was performed usingAnti-FLAG® M2 Magnetic Beads (Sigma-Aldrich Corporation, MO) accordingto the manufacturer's protocol with modifications. Typically this steprequires overnight incubation of the protein materials with the beads;however, by increasing the ratio of beads to protein, the bindingconditions were optimized and the incubation time reduced to 4 hrs, suchthat almost all available protein was bound to the beads (FIGS. 9Athrough 9D). Thus, the magnetic beads were prepared as per themanufacturer's protocol and mixed with the CFPS sample at 1:10 (v/v).Briefly, 5× packed bead volume TBS (50 mM Tris HCl, 150 mM NaCl, pH 7.4)was added and mixed thoroughly with appropriate bead volume. The tubewas placed on a magnetic rack and the supernatant was removed followedby one more wash with 5× volume TBS. Lysate was then added to the beadsand diluted 3 times with Tris-buffered saline (TBS) (50 mM Tris-HCl, 150mM NaCl, pH 7.4) in the presence of Tween-20 (0.1%) and, in someembodiments, a protease inhibitor cocktail (Roche cOmplete™ ProteaseInhibitor Cocktail Tablets). After incubation and binding at roomtemperature (≥4 hrs), the affinity captured material was isolated bymagnetic bead separation on a magnetic rack and the beads were washedthree times with 10 times bead volume TBS. Elution of FLAG™-tag proteinwas performed using 5× bead volume 0.1M Glycine pH 3.0. The supernatantcontaining the FLAG™-protein was transferred to a vial containingTris-HCl pH 8.0 for re-equilibration to neutral pH according to themanufacturer's recommendations (Sigma-Aldrich Corporation, MO).

Experiments were also performed to optimize the Enterokinase treatmentconditions. Briefly, to remove the FLAG™-tag, eluted FLAG™-proteinsamples were adjusted to a final concentration of 50 mM NaCl and 2 mMCaCl₂), followed by addition of Enterokinase (Novagen, Inc., WI) (1 unitof recombinant Enterokinase per 100 ng FLAG™-protein), and incubation ofthe mixture at room temperature for 4 hours. Under these conditions,complete cleavage of the FLAG™ epitope from the tagged EPO protein wasobserved (FIG. 9B). Following cleavage, Enterokinase was removed viaaffinity-based capture using agarose matrix embedded with soybeantrypsin inhibitor according to the manufacturer's protocol (EKapture™Agarose, Novagen, Inc., MI) according to the manufacturer's protocol.The efficiency of the Enterokinase capture step was assessed using anEnterokinase-specific activity assay (SensoLyte Rh110, AnaSpec, Inc.,CA); no observable enzymatic activity remained in the EPO purifiedsample (FIG. 9C). The de-tagged protein was purified using a 2 mlSephacryl 5200 HR (GE Healthcare Bio-Sciences, PA) size exclusionchromatography column (ratio 20:1, flow rate of 35 μl/min). Proteinsamples were analyzed in 4-12% Bis-Tris SDS-PAGE gels with SimplyBlueSafeStain (Life Technologies, Corporation, CA) according to themanufacturer's recommendations. Western blot analyses were performed andpreliminary assessment of protein yields during the purification processwas performed using yeast cell-free protein synthesis systems spiked inwith known amounts of FLAG-EPO followed by protein purification usingthe resins and buffers described above. Overall purification yields forEPO indicated protein recovery at an estimated 50±5% (FIG. 9A). Thus,the presently disclosed process allows generation of single dose amountsof protein products from small cell free reaction volumes (1-5 ml).

Example 9 Activity of Human Erythropoietin Purified from a YeastCell-Free Protein Synthesis System

EPO was expressed in a PDMS-based CECF reactor hosting a yeast CFPSsystem and purified using the FLAG™-affinity chromatography-basedprotocol as described above. A standard EPO-dependent cell viabilityassay was employed to determine the bioactivity of the purified protein.This assay is based on quantitation of luminescent signal generated inthe presence of ATP, which is directly proportional to the number ofmetabolically active cells present in the well. Briefly, TF-1 cells(ATCC # CRL 2003) were maintained in RPMI 1640 (ATCC 30-2001) mediasupplemented with 10% Fetal Bovine Serum, Penicillin/Streptomycin (100U/ml), 2 ng/ml recombinant Human GM-CSF and grown at 37° C. 5% CO₂.Cells were washed three times in growth medium without GM-CSF and platedin 96-well culture plates (2×10⁴-1×10⁵ cells/ml) in the presence ofserial dilutions of sample protein or controls (final volume 100μl/well). Cells were then incubated at 37° C. for 48-72 hours. At theend of the incubation period, cells were removed from 37° C. and allowedto equilibrate at room temperature for 30 minutes. An equal volume (100μl) of room-temperature CellTiter-Glo® reagent (Promega) was added tothe cells followed by shaking of the plates at 900 rpm for 2 minutes.After incubation of the plates for 10 minutes at room temperature,luminescence was measured. The results showed that end-to-end producedEPO has activity comparable to that of a commercially available anddeglycosylated EPO protein (FIG. 10). The EC50 was calculated at 7.469for the purified EPO expressed in yeast, whereas the EC50 for the EPOcontrol was 3.363.

Example 10 Bacterial Cell-Free Protein Synthesis

Bacterial S30 crude extracts were generated from a genomically recodedrelease factor 1 (RF1) deficient E. coli strain (E. coli C321.4Δ.705)(Lajoie, et al., 2013, Science, 342:357-360) and described in detail in(Kwon and Jewett, 2015, Sci. Rep. 5, DOI:10.1038/srep08663). Thebacterial combined transcription/translation protein synthesis reactionwas performed in the PDMS reactor under a batch format and had thefollowing final composition: 57 mM HEPES, pH 7.4, 12 mM magnesiumglutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 1.2 mMATP, 0.85 mM CTP, 0.85 mM GTP, 0.85 mM UTP, 0.034 mg/ml folinic acid,0.171 mg/ml tRNA, 2 mM of each of the 20 amino acids, 33.3 mMphosphoenol pyruvate, 0.33 mM NAD, 0.27 mM coenzyme A, 4 mM oxalic acid,1 mM putrescine, 1.5 mM spermidine, 4 mM glutathione disulfide, 1 mMglutathione, 100 μg/ml disulfide bond isomerase DsbC (Enzo LifeSciences, Inc., NY), 0.1% Brij-35, 1.3 U/μl T7 Polymerase (Thermo FisherScientific, MA), 13.3 ng/μl plasmid, and 15% (v/v) E. coli extract. Thenecessity of addition of exogenous tRNA to the bacterial CFPS reactionswas shown previously (Kim, et al., 2006, J. Biotechnol. 126:554-561).Prior to use, E. coli extracts were treated with 1 mM iodoacetamide(IAM) (Sigma-Aldrich Corporation, MO) at room temperature for 30 min.The reaction solution was loaded into the reactor and incubated at 30°C. for 10 hours. The solubility of the expressed rhGM-CSF protein wasassessed as previously described. Initial assessment of protein yieldsshowed that the bacterial CFPS system had variability of approximately13% between different lysate lots, whereas variability between replicateexperiments using the same lysate lot on different days was less than5%.

Quantitative assessment of the cell-free rhEPO and rhGM-CSF products wasperformed using Western blot analysis in the presence of known amountsof protein standards and/or the WES™ system (ProteinSimple, CA). ForrhEPO, Western blot analyses were performed using a rabbit anti-EPOantibody (H-162, Santa Cruz Biotechnology, Inc., TX) and a horseradishperoxidase (HRP) conjugated goat anti-rabbit secondary antibody(111-035-003, Jackson ImmunoResearch Laboratories, Inc., PA). ForrhGM-CSF, Western blot analyses were performed using a rabbitanti-GM-CSF antibody (AbCam, PLC, MA) and an HRP conjugated goatanti-rabbit secondary antibody (111-035-003, Jackson ImmunoResearchLaboratories, Inc., PA). In both cases, proteins were analyzed using aStorm 840 PhosphorImager (GE Healthcare Bio-Sciences, PA).

Using these conditions, GM-CSF constructs carrying signal peptidesequences upstream of the coding region were expressed in the bacterialCFPS system. Western blot analysis of the GM-CSF product showed that theexpressed protein was present almost exclusively in a soluble, oxidizedform (FIG. 11). Importantly, the nascent peptide was accuratelyprocessed in the lysate generating mature protein having a sequence thatwas identical to that of the pharmaceutical product (Sargramostim™, arecombinant GM-CSF marketed by Genzyme under the tradename Leukine™). Tothis end, LC-MS/MS confirmed expected target sequences for GM-CSFproduced in the bacterial system (102 of 127 amino acids identified(80.3% coverage)), and confirmed the presence of the correct N-terminalsequence in the protein product as the result of accurate cleavage ofthe signal peptide sequence from nascent polypeptide.

Using the bacterial CECF protein synthesis system, human GM-CSF wasproduced at ˜2 mg per ml of reaction. Reactions volumes in this systemcan be scaled up through the use of larger reactors and/orparallelization to allow production of greater amounts and/or highertherapeutic doses of protein biologics, as needed.

Example 11 Purification of Human GM-CSF from a Bacterial Cell-FreeProtein Synthesis System

A modular strategy was developed for continuous flow purification ofGM-CSF from bacterial cell-free systems using a combination of IonEXchange (IEX) and Size Exclusion Chromatography (SEC) steps (FIG. 8B)similar to the method used by Zawada et al., 2011, Biotechnol. Bioeng.108:1570-1578. Briefly, bacterial CFPS samples (1 ml) diluted 1:1 with10 mM sodium phosphate, pH 6.5, during loading (flow rate of 250 μl/min)onto a 5 cm×1.25 cm (ID) Diethylaminoethyl (DEAE)-Sepharose Fast Flowresin (˜6 ml bed volume) (GE Healthcare Bio-Sciences, PA) equilibratedat 10 mM sodium phosphate, pH 6.5. The column was washed and sampleswere eluted (flow rate of 500 μl/min) with a step gradient usingincreasing concentrations of NaCl (0 to 1M) in the same sodium phosphatebuffer. Western blot analysis showed that almost all of the GM-CSFprotein was recovered upon elution with 200 mM NaCl. The entire 200 mMsalt fraction was collected, dialyzed and concentrated over aregenerated cellulose 3 kDa molecular weight cut-off membrane (Ultracel,Merk EMD Millipore, MA). Sample fractions containing GM-CSF were pooled,dialyzed, the concentrated samples were loaded on a 6 ml Sephacryl® S200HR (GE Healthcare Bio-Sciences, PA) size exclusion chromatography column(40:1 length:width, column flow rate of 18-35 μl/min) and developed at aflow rate of 20 μl/min. Total protein in fractionation samples wasquantified using a Pierce™ BCA protein assay (Life TechnologiesCorporation, CA) and GM-CSF purity was assessed in 4-12% SDS-PAGE gelswith SimplyBlue SafeStain™ (Life Technologies Corporation, CA) accordingto the manufacturer's recommendations. The Coomassie blue stainingprotein profiles of the IEX and SEC column samples are shown in FIG. 12.Purification of GM-CSF produced in the bacterial cell-free system wascompleted within 8 hours. Protein quantitative assessment indicated thatthe overall rhGM-CSF recovery was at an estimated 20±3%, thus allowinggeneration of single dose amounts of protein products from small cellfree reaction volumes (1-5 ml).

Example 12 Activity of Human GM-CSF Purified from a Bacterial Cell-FreeProtein Synthesis System

GM-CSF was synthesized in a bacterial lysate hosted in a PDMS reactorand purified using the aforementioned fluidic ion exchange and sizeexclusion chromatography methods. The biological activity of thepurified product was evaluated using a standard cell-based assay thatmonitors the ability of the target protein to induce proliferation of aGM-CSF-depended human cell line, TF-1. The bioactivity assay wasperformed as described above. End-to-end produced GM-CSF was found tohave similar activity to that of a commercially available controlprotein (FIG. 13). The EC50 was calculated at 0.01823 for the purifiedGM-CSF expressed in the bacterial cell-free protein synthesis system,whereas the EC50 for the GM-CSF control was 0.02407.

Example 13 An Affinity FLAG™-Tag Approach for GM-CSF Purification from aBacterial Cell-Free Protein Synthesis System

To demonstrate the system's flexibility, an affinity-based approach forGM-CSF purification from the bacterial platform was developed, similarto the approach described above for EPO purification from the yeastplatform. To this end, a construct encoding GM-CSF carrying a FLAG™-tagwas generated and subjected to coupled transcription/transcription asdescribed above. The synthesized protein was found in the solublefraction of the reaction (FIG. 14). Protein was purified using theanti-FLAG™ affinity chromatography method and its bioactivity wasassessed using the GM-CSF-depended human cell line bioassay as describedabove. GM-CSF product was found to have similar activity to that of acommercially available control protein (FIG. 15).

Example 14 Expression, Purification, Posttranslational Modifications,and Activity of Human EPO Produced in a Bacterial Cell-Free ProteinSynthesis System

To further assess the flexibility of the developed platform, human EPOwas expressed in bacterial CFPS system and purified using theaforementioned FLAG™-Tag affinity chromatography approach. LC-MS/MSconfirmed human EPO sequence expressed in the bacterial CFPS system((123 of 166 amino acids were identified (74% coverage)). The biologicalactivity of bacterially expressed and purified EPO was evaluated using astandard cell-based assay that monitors the ability of the targetprotein to induce proliferation of an EPO-dependent human cell line,TF-1. The results showed that the activity of the protein product wasnearly identical to that of a commercial control EPO protein (FIG. 16).

To evaluate the ability of the bacterial CFPS platform to conferhuman-like glycosylation of target proteins, a heterologouscross-kingdom reconstituted system consisting of the bacterial cell-freetranslation system and the mammalian translocation machinery wasdeveloped. To this end, mammalian-derived SRP, ER and Golgi componentswere isolated from CHO cells using previously described methods (e.g.,Walter and Blobel, 1983, Methods Enzymol. 96:84-93) and added to abacterial CFPS system programmed for EPO expression. FIG. 17 shows theability of the heterologous reconstituted system to generate EPO proteinproducts having a MW that is similar to that of the glycosylated EPOcontrol protein. Gel samples containing the high MW EPO band wereisolated and subjected to Mass Spectrometry analysis; LC-MS analysisconfirmed EPO present in the high MW band.

Example 15 Rapid Cell-Free Expression and Purification of Active rhEPO

Enhancement of rhEPO Expression in a Yeast Cell Free System ThroughAlleviation of Substrate Limitations and mRNA Translation InhibitoryFactors

One objective was to enhance product yields and functional activity ofrhEPO expressed in a Saccharomyces cerevisiae-based CFPS system. Thiswas achieved by a two-fold approach focusing on: a) alleviating theexhaustion of essential substrates (e.g., nucleotide triphosphates andamino acids) that are consumed during protein synthesis and removal oftoxic byproducts (e.g., inorganic phosphates) accumulated during proteinsynthesis (Spirin 2004, Trends Biotechnol. 22:538-545; Schoborg et al.,2014, Biotechnol J. 9:630-640) and b) eliminating translation initiationinhibitory features, such as canonical and non-canonical initiationcodons contained within the coding sequence that cause ribosomaldiversion and consequently reduction in the synthesis of the correct,full-length protein product (Matsuda and Mauro, 2010, PLoS One, 5, DOI:10.1371/journal.pone.0015057; Chappell et al., 2006, Proc. Natl. Acad.Sci. U.S.A. 103:18077-18082). The former approach involved adaptation ofa CECF protein synthesis format that has broadly been utilized forgeneration of highly productive CFPS systems (Shirokov et al., 2007,Methods Mol. Biol. 375:19-55). The latter approach involved the use ofsynonymous mutations to either remove alternative initiation codons ordecrease their utilization contained within the natural EPO signalpeptide sequences. Signal peptides are short leader peptides found onthe N-terminus of proteins destined for the secretory pathway. Signalpeptides are portable, i.e., they can function on different genes, andare cleaved from the nascent polypeptide to generate the mature protein.To this end, EPO constructs carrying recoded signal peptide sequencesupstream of the coding region as well as a FLAG™-Tag sequence betweenthe signal peptide and the coding region were generated and subjected tocombined transcription/translation in the yeast CECF protein synthesissystem (FIG. 6). Using the yeast CECF protein synthesis system, rhEPOwas produced at approximately 40 μg per ml of reaction with the majorityof the expressed protein being present in the soluble fraction of theCFPS reaction mixture (FIG. 6).

A Modular Fluidic Process for Rapid Purification of Cell-Free ExpressedrhEPO

FIG. 6 presents a schematic diagram of the construct used for cell-freeexpression of rhEPO in the yeast CECF protein synthesis system. The PCRtemplate for rhEPO contains a T7 promoter (T7), translational enhancer(E), signal peptide (SP), and three FLAG™ epitopes, with an enterokinasecleavage site upstream of the mature coding sequence. FIG. 6 also showsa Western blot analysis of rhEPO expression in a yeast cell-free proteinsynthesis system at 22° C. As can be observed in FIG. 6, the majority ofrhEPO expressed in the yeast CFPS system is present in the solublefraction.

rhEPO expressed in the yeast CFPS system was purified according to thestrategy described in FIG. 8A. The first step of the purificationprocess involved separation of the expressed protein from the yeastlysate using anti-FLAG™ affinity chromatography. Typically this steprequires overnight incubation of the protein materials with the beads;however, upon optimization of the binding conditions (increasing theratio of beads to protein) it was possible to reduce this time to 4hours, during which time almost all available protein was bound to thebeads (FIG. 9A).

The next step included elution of the protein off the beads under acidicconditions (pH 3) (FIG. 9A). Treatment of the eluted protein withEnterokinase resulted in cleavage of the FLAG™ epitope from the taggedEPO protein as demonstrated by the reduction in the molecular weight ofthe protein product (Western blot using anti-EPO antibody, FIG. 9A) andthe disappearance of the FLAG™ epitope (Western blot using anti-FLAG™antibody, FIG. 9B). Enterokinase was then removed via the use of anagarose matrix embedded with soybean trypsin inhibitor that specificallybinds to the enzyme. An Enterokinase-specific activity assay showed thatthis affinity-based enzyme capture step resulted in removal of theEnterokinase from the protein sample (FIG. 9C). The protein product wasvisualized using a Coomassie blue staining gel (FIG. 9D).

Western blot analysis (anti-EPO) of FLAG™-rhEPO expressed in a yeastcell-free protein synthesis system during the various steps of proteinpurification. (FIG. 9B). Western blot analysis (anti-FLAG™) of EPOsample before and after Enterokinase treatment for FLAG™-tag removal.(FIG. 9C) The efficiency of the Enterokinase capture and removal stepassessed using a fluorogenic substrate-based enzymatic assay; uponenzyme cleavage, this substrate generates a rhodamine fluorophore thatis detected at excitation/emission=490/520 nm. (FIG. 9D) Coomassie bluestaining of protein samples before and after FLAG-affinitychromatography.

Mass spectrometry analysis showed that the nascent FLAG™-rhEPOpolypeptides synthesized in vitro were accurately processedenzymatically, generating mature rhEPO protein with a sequence that wasidentical to that of the pharmaceutical product (Epogen, Amgen, Inc.,CA). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) usingtrypsin and Glu-C digests, as well as the overlap between them,confirmed the expected target sequences [86.7% coverage (144 of 166amino acids) for rhEPO produced in the yeast system] as well as thepresence of the correct N-terminal sequence in the rhEPO product as theresult of enzymatic cleavage of the FLAG™ tag during the proteinpurification process (see FIGS. 18-20).

Production of Bioactive rhEPO

A standard EPO-dependent cell viability assay was employed to determinethe bioactivity of the purified protein. This assay is based onquantitation of luminescent signal generated in the presence of ATP,which is directly proportional to the number of metabolically activecells present in the well. The results showed that produced EPO hasactivity that is comparable to that of a commercially available EPOprotein (FIG. 10).

Biological activity of rhEPO synthesized in a PDMS-based reactor hostinga yeast CFPS system under continuous exchange format and purified asdescribed in FIG. 8A. Protein biological activity was assessed using astandard human TF-1 cell-based proliferation assay (FIG. 10). Shown arerepresentative results from two biological replicates at multiple rhEPOprotein concentrations. Data represent the mean±SD of luminescencerelative light units produced in the biological experiments.

Example 16 Rapid Cell Free Expression and Purification of ActiverhGM-CSF

Alleviation of mRNA Translation Inhibitory Features and RedoxEnvironment Optimization Increases Expression of Oxidized rhGM-CSF in aBacterial Cell Free System

An assay optimization approach was used to maximize expression offunctionally folded human GM-CSF in the bacterial batch CFPS system.Similarly to the aforementioned yeast expression strategy, recodedsignal peptides were generated and cloned upstream of the rhGM-CSFcoding region to eliminate inhibitory features, such as secondary startsites, and consequently increase the productive ribosomal recruitment tothe main mRNA initiation codon in the bacterial cell-free proteinsynthesis system (FIG. 11). Additionally, cell-free reactions wereperformed in an oxidizing environment that favors disulfide bondformation (GM-CSF contains two intramolecular disulfide bonds in itsnative form) and supplemented with specific foldases and chaperonemolecules, conditions that have been shown to allow for correct proteinfolding and synthesis of soluble and active target proteins (Goerke andSwartz, 2008, Biotechnol. Bioeng. 99:351-367). This approach included:a) iodoacetamide (IAM) pre-treatment of cell extracts (IAM inactivatesreducing activity in lysate by inactivating disulfide-reducing enzymes,such as, for example, thioredoxin reductase), b) adjustment of the redoxpotential by adding an oxidized glutathione buffer (“GSH/GSSG”containing glutathione (GSH) and glutathione disulfide (GSSG)), and c)addition of an E. coli periplasmic disulfide isomerase (DsbC) tofacilitate formation of correct disulfide bonds (incorrect disulfidebonds may lead to poor protein folding and aggregation of misfoldedproteins) (Goerke and Swartz, 2008, Biotechnol. Bioeng. 99:351-367).Addition of nonionic detergents has also been shown to prevent productaggregation and improve solubility of proteins expressed in CSFP systems(Shirokov et al., 2007, Methods Mol. Biol. 375:19-55). Accordingly,Brij® 35 was included in the CFPS system. Using these conditions,rhGM-CSF protein was expressed from the aforementioned constructs atapproximately 2 mg per ml of reaction and found to be present almostexclusively in a soluble, oxidized form (see FIG. 11). FIG. 11 shows aschematic diagram of the construct used for cell-free expression ofrhGM-CSF in the bacterial cell-free protein synthesis system. Theplasmid template for rhGM-CSF contains a T7 promoter (T7), translationalenhancer (E), and signal peptide (SP) upstream of the mature codingsequence. FIG. 11 also shows a Western blot analysis of cell-freeexpressed rhGM-CSF in a bacterial lysate. CFPS reaction was performed inthe presence of nonionic detergent Brij 35 (0.1%), IAM and DsbC at 30°C.

A Modular Fluidic Process for Rapid Purification of Bacterial Cell-FreeExpressed rhGM-CSF

rhGM-CSF expressed in the CFPS system was subjected to a proteinpurification scheme involving ion exchange and size exclusionchromatography (FIG. 8B), similar to the protocol described earlier(Zawada et al., 2011, Biotechnol. Bioeng. 108:1570-1578). Bacterial CFPSsamples were loaded onto the ion exchange module and subjected toelution with increasing concentrations of NaCl. The 200 mM saltfractions containing rhGM-CSF were pooled, dialyzed, concentrated, andloaded on a size exclusion chromatography module.

The Coomassie blue staining profiles of specific ion exchange and sizeexclusion samples during the various steps of the purification processare shown in FIG. 12, indicating the isolation of a protein with theexpected molecular weight of rhGM-CSF. Commercial human GM-CSF was usedas a control. LC-MS/MS analysis also confirmed the production of theexpected target sequence [80.3% coverage (102 of 127 amino acids)] forthe rhGM-CSF protein expressed in the bacterial system as well asestablished the presence of the correct N-terminal sequence in theprotein product as the result of accurate cleavage of the signal peptidesequence from the nascent polypeptide in the bacterial lysate (see FIGS.21-23). The correct cleavage of the in vitro expressed rhGM-CSF proteinis most likely due to signal peptidase activity present in the bacterialextract used in the CFPS system (Ahn et al., 2007, Nucleic Acids Res.35, DOI: 10.1093/nar/gkl917).

Production of Bioactive rhGM-CSF

The biological activity of the expressed and purified rhGM-CSF product wsynthesized in a PDMS-based reactor hosting a bacterial CFPS systemunder batch format and purified as described in FIG. 8B as evaluatedusing a standard cell-based assay that monitors the ability of thetarget protein to induce proliferation of a GM-CSF-dependent human cellline, TF-1. Protein biological activity was assessed using a standardhuman TF-1 cell-based proliferation assay (FIG. 13). Shown arerepresentative results from two biological replicates at multiplerhGM-CSF protein concentrations. Data represent the mean±SD ofluminescence relative light units produced in the biologicalexperiments. Purified GM-CSF was found to have similar activity to thatof a commercially available control protein (FIG. 13).

Additional approaches for in vitro protein glycosylation in both theyeast and bacterial CFPS systems are being tested, involving a)incorporation of non-genetically encoded amino acids for site specificglycoform bioconjugation and b) in vitro enzymatic glycosylation. Suchapproaches may result in the generation of chemically homogeneousglycoproteins in the bacterial or yeast CFPS systems.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

1. A cell-free method for producing an amount of a protein biologicformulation at pharmaceutical dosage levels at a point of need, themethod comprising: loading by a loading module a reaction solutionincluding one or more reaction reagents to a reactor module; loading bya feeder module a feeding solution to the reactor module, the reactormodule having a first chamber and a second chamber divided by amembrane; receiving at the first chamber the one or more reaction agentsfrom the loading module; receiving at the second chamber the feedingsolution from the feeder module; producing by the reactor module atarget protein via a process selected from the group consisting of: (i)continuous exchange cell-free (CECF) and (ii) batch protein synthesis;separating by a chromatography module the target protein from one ormore components of the reaction solution; and formulating by a finishingmodule the target protein; wherein the producing, separating andformulating of the target protein are completed within a period of 24hours.
 2. The method of claim 1, wherein: the producing, separating andformulating of the target protein are completed within a period of 12hours.
 3. The method of claim 1, wherein: the producing, separating andformulating of the target protein are completed within a period of 10hours.
 4. The method of claim 1, wherein the target protein is producedin an amount between about 40 μg/ml and about 8 mg/ml protein.
 5. Themethod of claim 1, further comprising: facilitating by a control systempurification of different proteins on the same platform via switchingamong various chromatography matrices.
 6. The method of claim 1, furthercomprising: removing via one or more sampling ports sample aliquots fromthe reaction chamber for assessment of protein synthesis and formulationprocesses.
 7. The method of claim 1, further comprising: directingfluidics between one or more of the loader, feeder, reactor,chromatography and finishing modules and facilitating addition andremoval of a solution, including the reaction solution and the feedingsolution.
 8. The method of claim 1, wherein: formulating includes sizeexclusion using a chromatography matrix.
 9. The method of claim 1,wherein: separating the target protein employs at least one of (a) tagbased affinity chromatography, (b) protease affinity chromatography, and(c) size exclusion chromatography steps, or a combination thereof. 10.The method of claim 1, wherein: separating the target protein employs atleast one of (a) ion-exchange chromatography, (b) protein concentration,and (c) size exclusion chromatography steps, or a combination thereof.11. A cell-free method for producing an amount of at least one proteinbiologic formulation at pharmaceutical dosage levels at a point of need,the method comprising: loading by a loading module a reaction solutionincluding one or more reaction reagents to a reactor module; loading bya feeder module a feeding solution to the reactor module, the reactormodule having a first chamber and a second chamber divided by amembrane; receiving at the first chamber the one or more reaction agentsfrom the loading module; receiving at the second chamber the feedingsolution from the feeder module; producing by the reactor module atarget protein via a process selected from the group consisting of: (i)continuous exchange cell-free (CECF) and (ii) batch protein synthesis;monitoring the produced target protein by one or more analytical modulesto asses target protein purity level; directing the produced targetprotein to one of multiple separation modules in accordance with targetprotein requirements; separating the target protein from one or morecomponents of the reaction solution; and formulating by a finishingmodule the target protein; wherein the producing, monitoring, directing,separating and formulating of the target protein are completed within aperiod of 24 hours.
 12. The method of claim 11, wherein: the producing,separating and formulating of the target protein are completed within aperiod of 12 hours.
 13. The method of claim 11, wherein: the producing,separating and formulating of the target protein are completed within aperiod of 10 hours.
 14. The method of claim 11, wherein monitoring theproduced target protein by one or more analytical modules includesremoving via one or more sampling ports sample aliquots from thereaction chamber.
 15. The method of claim 1, wherein the target proteinis produced in an amount between about 40 μg/ml and about 8 mg/mlprotein.
 16. The method of claim 1, further comprising: facilitating bya control system purification of different proteins on the same platformvia switching among various chromatography matrices.
 17. The method ofclaim 1, further comprising: directing fluidics between one or more ofthe loader, feeder, reactor, chromatography and finishing modules andfacilitating addition and removal of a solution, including the reactionsolution and the feeding solution.
 18. The method of claim 1, wherein:formulating includes size exclusion using a chromatography matrix. 19.The method of claim 1, wherein: separating the target protein employs atleast one of (a) tag based affinity chromatography, (b) proteaseaffinity chromatography, and (c) size exclusion chromatography steps, ora combination thereof.
 20. The method of claim 1, wherein: separatingthe target protein employs at least one of (a) ion-exchangechromatography, (b) protein concentration, and (c) size exclusionchromatography steps, or a combination thereof.